Novel compounds

ABSTRACT

Compounds of formula (I) 
     
       
         
         
             
             
         
       
     
     wherein R 1  is 2-indanyl, R 2  is 1-methylpropyl, R 3  is a group selected from 2,6-dimethyl-3-pyridyl or 4,6-dimethyl-3-pyridyl, R 4  represents methyl and R 5  represents hydrogen or methyl or, R 4  and R 5  together with the nitrogen atom to which they are attached represent morpholino and pharmaceutically acceptable derivatives thereof are described, as are processes for their preparation, pharmaceutical compositions containing them and their use in medicine, particularly their use as oxytocin antagonists.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/451,196 filed Apr. 19, 2012; U.S. application Ser. No. 13/035,366filed Feb. 25, 2011, issued as U.S. Pat. No. 8,202,864; which is acontinuation of U.S. application Ser. No. 12/850,386 filed Aug. 4, 2010,issued as U.S. Pat. No. 7,919,492; which is a continuation of U.S.application Ser. No. 12/480,182 filed Jun. 8, 2009, now abandoned; whichis a continuation of U.S. application Ser. No. 11/630,179 filed Dec. 19,2006, issued as U.S. Pat. No. 7,550,462; which was filed pursuant to 35U.S.C. §371 as a U.S. National Phase Application of InternationalApplication No. PCT/EP2005/006760 filed Jun. 21, 2005; which claimspriority to Great Britain Application No. 0414093.5, filed Jun. 23,2004.

FIELD OF THE INVENTION

This invention relates to novel diketopiperazine derivatives having apotent and selective antagonist action at the oxytocin receptor, toprocesses for their preparation, pharmaceutical compositions containingthem and to their use in medicine.

The hormone oxytocin is a potent contractor of the uterus and is usedfor the induction or augmentation of labour. Also the density of uterineoxytocin receptors increases significantly by >100 fold during pregnancyand peaks in labour (pre-term and term).

Pre-term births/labour (between 24 and 37 weeks) causes about 60% ofinfant mortality/morbidity and thus a compound which inhibits theuterine actions of oxytocin e.g. oxytocin antagonists, should be usefulfor the prevention or control of pre-term labour.

International patent application WO 99/47549 describes diketopiperazinederivatives including 3-benzyl-2,5-diketopiperazine derivatives asinhibitors of fructose 1,6-bisphosphate (FBPase). International patentapplication WO 03/053443 describes a class of diketopiperazinederivatives which exhibit a particularly useful level of activity asselective antagonists at the oxytocin receptor. A preferred class ofcompounds described therein is represented by the formula (A)

Such compounds include those wherein inter alia R₁ is 2-indanyl, R₂ isC₃₋₄alkyl, R₃ is a 5 or 6 membered heteroaryl group linked to the restof the molecule via a carbon atom in the ring, R₄ represents the groupNR₅R₆ wherein R₅ and R₆ each represent alkyl e.g. methyl, or R₅ and R₆together with the nitrogen atom to which they are attached form a 3 to 7membered saturated heterocyclic ring which heterocycle may contain anadditional heteroatom selected from oxygen.

International patent application WO 2005/000840 describesdiketopiperazine derivatives of formula (B)

wherein R₁ is 2-indanyl, R₂ is 1-methylpropyl, R₃ is2-methyl-1,3-oxazol-4-yl and R₄ and R₅ together with the nitrogen atomto which they are attached represent morpholino.

We have now found a novel group of selective oxytocin receptorantagonists which exhibit a particularly advantageous pharmacokineticprofile.

The present invention thus provides at least one chemical entityselected from compounds of formula (I)

wherein R₁ is 2-indanyl, R₂ is 1-methylpropyl, R₃ is a group selectedfrom 2,6-dimethyl-3-pyridyl or 4,6-dimethyl-3-pyridyl, R₄ representsmethyl and R₅ represents hydrogen or methyl or R₄ and R₅ together withthe nitrogen atom to which they are attached represent morpholino andpharmaceutically acceptable derivatives thereof.

It will be appreciated that the compounds of formula (I) possess theabsolute stereochemistry depicted at the asymmetric carbon atoms bearinggroups R₁, R₂ and R₃, i.e. the stereochemistry at these positions isalways (R). Nevertheless, it should also be appreciated that althoughsuch compounds are substantially free of the (S)-epimer at each of R₁,R₂ and R₃, each epimer may be present in small amounts, for example 1%or less of the (S)-epimer may be present.

It will also be appreciated that the group R₂ contains an asymmetriccarbon atom and that the invention includes both the (R)- and(S)-epimers thereof.

In one embodiment of the invention, R₂ is (1S)-1-methylpropyl. Inanother embodiment of the invention, R₂ is (1R)-1-methylpropyl.

One embodiment of the invention is the compounds the preparation ofwhich is specifically described in examples 1, 2, 3 and 6. Anotherembodiment of the invention is the compounds the preparation of which isspecifically described in examples 1, 2 and 3. A further embodiment ofthe invention is the compounds the preparation of which is specificallydescribed in examples 3 and 6. A yet further embodiment of the inventionis the compound the preparation of which is specifically described inexample 3. Another embodiment of the invention is the compound thepreparation of which is specifically described in example 1.

In one aspect, chemical entities useful in the present invention may beat least one chemical entity selected from:

-   (2R)-2-{(3R,6R)-3-(2,3-dihydro-1H-inden-2-yl)-6-[(1S)-1-methylpropyl]-2,5-dioxo-1-piperazinyl}-2-(2,6-dimethyl-3-pyridinyl)-N-methylethanamide;-   (2R)-2-{(3R,6R)-3-(2,3-dihydro-1H-inden-2-yl)-6-[(1S)-1-methylpropyl]-2,5-dioxo-1-piperazinyl}-2-(2,6-dimethyl-3-pyridinyl)-N,N-dimethylethanamide;-   (3R,6R)-3-(2,3-dihydro-1H-inden-2-yl)-1-[(1R)-1-(2,6-dimethyl-3-pyridinyl)-2-(4-morpholinyl)-2-oxoethyl]-6-[(1S)-1-methylpropyl]-2,5-piperazinedione;-   (2R)-2-{(3R,6R)-3-(2,3-Dihydro-1H-inden-2-yl)-6-[(1S)-1-methylpropyl]-2,5-dioxo-1-piperazinyl}-2-(4,6-dimethyl-3-pyridinyl)-N,N-dimethylethanamide;-   (2R)-2-{(3R,6R)-3-(2,3-Dihydro-1H-inden-2-yl)-6-[(1S)-1-methylpropyl]-2,5-dioxo-1-piperazinyl}-2-(4,6-dimethyl-3-pyridinyl)-N-methylethanamide;    (3R,6R)-3-(2,3-dihydro-1H-inden-2-yl)-1-[(1R)-1-(4,6-dimethyl-3-pyridinyl)-2-(4-morpholinyl)-2-oxoethyl]-6-[(1S)-1-methylpropyl]-2,5-piperazinedione;    and pharmaceutically acceptable derivatives thereof.

As used herein, the term “pharmaceutically acceptable” means a compoundwhich is suitable for pharmaceutical use. Salts and solvates ofcompounds of the invention which are suitable for use in medicine arethose wherein the counterion or associated solvent is pharmaceuticallyacceptable. However, salts and solvates having non-pharmaceuticallyacceptable counterions or associated solvents are within the scope ofthe present invention, for example, for use as intermediates in thepreparation of other compounds of the invention and theirpharmaceutically acceptable salts and solvates.

As used herein, the term “pharmaceutically acceptable derivative”, meansany pharmaceutically acceptable salt, solvate, or prodrug e.g. ester, ofa compound of the invention, which upon administration to the recipientis capable of providing (directly or indirectly) a compound of theinvention, or an active metabolite or residue thereof. Such derivativesare recognizable to those skilled in the art, without undueexperimentation. Nevertheless, reference is made to the teaching ofBurger's Medicinal Chemistry and Drug Discovery, 5th Edition, Vol 1:Principles and Practice, which is incorporated herein by reference tothe extent of teaching such derivatives. In one aspect, pharmaceuticallyacceptable derivatives are salts, solvates, esters, carbamates andphosphate esters. In another aspect, pharmaceutically acceptablederivatives are salts, solvates and esters. In a further aspect,pharmaceutically acceptable derivatives are salts and solvates. Inanother aspect, pharmaceutically acceptable derivatives arephysiologically acceptable salts.

Suitable physiologically acceptable salts of compounds of the presentinvention include acid addition salts formed with physiologicallyacceptable inorganic acids or organic acids. Examples of such acidsinclude hydrochloric acid, hydrobromic acid, nitric acid, phosphoricacid, sulphuric acid, sulphonic acids e.g. methanesulphonic,ethanesulphonic, benzenesulphonic and p-toluenesulphonic, citric acid,tartaric acid, lactic acid, pyruvic acid, acetic acid, succinic acid,fumaric acid and maleic acid.

The present invention also relates to solvates of the compounds offormula (I), for example hydrates, or solvates with pharmaceuticallyacceptable solvents including, but not limited to, alcohols, for exampleethanol, iso-propanol, acetone, ethers, esters, e.g. ethyl acetate.

The compounds of the invention may also be used in combination withother therapeutic agents. The invention thus provides, in a furtheraspect, a combination comprising a compound of the invention or apharmaceutically acceptable derivative thereof together with a furthertherapeutic agent.

When a compound of the invention or a pharmaceutically acceptablederivative thereof is used in combination with a second therapeuticagent active against the same disease state the dose of each compoundmay differ from that when the compound is used alone. Appropriate doseswill be readily appreciated by those skilled in the art. It will beappreciated that the amount of a compound of the invention required foruse in treatment will vary with the nature of the condition beingtreated and the age and the condition of the patient and will beultimately at the discretion of the attendant physician or veterinarian.The compounds of the present invention may be used in combination withtocolytics or prophylactic medicines. These include, but are not limitedto, beta-agonists such as terbutaline or ritodrine, calcium channelblockers, e.g. nifedepine, non-steroidal anti-inflammatory drugs, suchas indomethacin, salts of magnesium, such as magnesium sulphate, otheroxytocin antagonists, such as atosiban, and progesterone agonists andformulations. In addition the compounds of the present invention may beused in combination with antenatal steroids including betamethasone anddexamethasone, prenatal vitamins especially folate supplements,antibiotics, including but not limited to ampicillin,amoxicillin/clavulanate, metronidazole, clindamycin, and anxiolytics.

The combinations referred to above may conveniently be presented for usein the form of a pharmaceutical formulation and thus pharmaceuticalformulations comprising a combination as defined above together with apharmaceutically acceptable carrier or excipient comprise a furtheraspect of the invention. The individual components of such combinationsmay be administered either sequentially or simultaneously in separate orcombined pharmaceutical formulations by any convenient route.

When administration is sequential, either the compound of the inventionor the second therapeutic agent may be administered first. Whenadministration is simultaneous, the combination may be administeredeither in the same or different pharmaceutical composition.

When combined in the same formulation it will be appreciated that thetwo compounds must be stable and compatible with each other and theother components of the formulation. When formulated separately they maybe provided in any convenient formulation, conveniently in such manneras are known for such compounds in the art.

The compounds of formula (I) have a high affinity for the oxytocinreceptors on the uterus of rats and humans and this may be determinedusing conventional procedures. For example the affinity for the oxytocinreceptors on the rat uterus may be determined by the procedure ofPettibone et al, Drug Development Research 30. 129-142 (1993). Thecompounds of the invention also exhibit high affinity at the humanrecombinant oxytocin receptor in CHO cells and this may be convenientlydemonstrated using the procedure described by Wyatt et al. Bioorganic &Medicinal Chemistry Letters, 2001 (11) p 1301-1305.

The compounds of the invention exhibit an advantageous pharmacokineticprofile including good bioavailability coupled with good aqueoussolubility. In one aspect, the compounds of the invention exhibit goodpotency and low intrinsic clearance. In another aspect, the compounds ofthe invention exhibit low intrinsic clearance.

The compounds of the invention are therefore useful in the treatment orprevention of diseases and/or conditions mediated through the action ofoxytocin. Examples of such diseases and/or conditions include pre-termlabour, dysmenorrhea, endometriosis and benign prostatic hyperplasia.

The compounds may also be useful to delay labour prior to electivecaesarean section or transfer of the patient to a tertiary care centre,treatment of sexual dysfunction (male and female), particularlypremature ejaculation, obesity, eating disorders, congestive heartfailure, arterial hypertension, liver cirrhosis, nephritic or ocularhypertension, obsessive-compulsive disorder and neuropsychiatricdisorders.

The compounds of the invention may also be useful for improvingfertility rates in animals, e.g. farm animals.

The invention therefore provides at least one chemical entity selectedfrom a compound of formula (I) and/or pharmaceutically acceptablederivatives thereof for use in therapy, particularly for use in humanand veterinary therapy, and in particular use as medicine forantagonising the effects of oxytocin upon the oxytocin receptor.

The invention also provides for the use of at least one chemical entityselected from a compound of formula (I) and/or pharmaceuticallyacceptable derivatives thereof for the manufacture of a medicament forantagonising the effects of oxytocin on the oxytocin receptor.

According to a further aspect, the invention also provides for a methodfor antagonising the effects of oxytocin upon the oxytocin receptor,comprising administering to a patient in need thereof an antagonisticamount of at least one chemical entity selected from a compound offormula (I) and/or pharmaceutically acceptable derivatives thereof.

It will be appreciated by those skilled in the art that reference hereinto treatment extends to prophylaxis as well as the treatment ofestablished diseases or symptoms.

It will further be appreciated that the amount of a compound of theinvention required for use in treatment will vary with the nature of thecondition being treated, the route of administration and the age and thecondition of the patient and will be ultimately at the discretion of theattendant physician. In general however doses employed for adult humantreatment will typically be in the range of 2 to 1000 mg per day,dependent upon the route of administration.

Thus for parenteral administration a daily dose will typically be in therange 2 to 50 mg, preferably 5 to 25 mg per day. For oral administrationa daily dose will typically be within the range 10 to 1000 mg, e.g. 50to 500 mg per day.

The desired dose may conveniently be presented in a single dose or asdivided doses administered at appropriate intervals, for example as two,three, four or more sub-doses per day.

While it is possible that, for use in therapy, a compound of theinvention may be administered as the raw chemical, it is preferable topresent the active ingredient as a pharmaceutical formulation.

The invention thus further provides a pharmaceutical formulationcomprising a compound of formula (I) and/or pharmaceutically acceptablederivatives thereof together with one or more pharmaceuticallyacceptable carriers thereof and, optionally, other therapeutic and/orprophylactic ingredients. The carrier(s) must be ‘acceptable’ in thesense of being compatible with the other ingredients of the formulationand not deleterious to the recipient thereof.

The compositions of the invention include those in a form especiallyformulated for oral, buccal, parenteral, inhalation or insufflation,implant, vaginal or rectal administration.

Tablets and capsules for oral administration may contain conventionalexcipients such as binding agents, for example, syrup, acacia, gelatin,sorbitol, tragacanth, mucilage of starch or polyvinylpyrrolidone;fillers, for example, lactose, sugar, microcrystalline cellulose,maize-starch, calcium phosphate or sorbitol; lubricants, for example,magnesium stearate, stearic acid, talc, polyethylene glycol or silica;disintegrants, for example, potato starch or sodium starch glycollate,or wetting agents such as sodium lauryl sulphate. The tablets may becoated according to methods well known in the art. Oral liquidpreparations may be in the form of, for example, aqueous or oilysuspensions, solutions emulsions, syrups or elixirs, or may be presentedas a dry product for constitution with water or other suitable vehiclebefore use. Such liquid preparations may contain conventional additivessuch as suspending agents, for example, sorbitol syrup, methylcellulose, glucose/sugar syrup, gelatin, hydroxyethylcellulose,carboxymethyl cellulose, aluminium stearate gel or hydrogenated ediblefats; emulsifying agents, for example, lecithin, sorbitan mono-oleate oracacia; non-aqueous vehicles (which may include edible oils), forexample, almond oil, fractionated coconut oil, oily esters, propyleneglycol or ethyl alcohol; solubilizers such as surfactants for examplepolysorbates or other agents such as cyclodextrins; and preservatives,for example, methyl or propyl p-hydroxybenzoates or ascorbic acid. Thecompositions may also be formulated as suppositories, e.g. containingconventional suppository bases such as cocoa butter or other glycerides.

For buccal administration the composition may take the form of tabletsor lozenges formulated in the conventional manner.

The composition according to the invention may be formulated forparenteral administration by injection or continuous infusion.Formulations for injection may be presented in unit dose form inampoules, or in multi-dose containers with an added preservative. Thecompositions may take such forms as suspensions, solutions, or emulsionsin oily or aqueous vehicles, and may contain formulatory agents such assuspending, stabilising and/or dispersing agents. Alternatively theactive ingredient may be in powder form for constitution with a suitablevehicle, e.g. sterile, pyrogen-free water, before use.

The compositions according to the invention may contain between 0.1-99%of the active ingredient, conveniently from 1-50% for tablets andcapsules and 3-50% for liquid preparations.

The advantageous pharmacokinetic profile of the compounds of theinvention is readily demonstrated using conventional procedures formeasuring the pharmacokinetic properties of biologically activecompounds.

The compounds of the invention and pharmaceutically acceptablederivatives thereof may be prepared by the processes describedhereinafter, said processes constituting a further aspect of theinvention. In the following description, the groups are as defined abovefor compounds of the invention unless otherwise stated.

Compounds of formula (I) may be prepared by reaction of the carboxylicacid (II), wherein R₁, R₂ and R₃ have the meanings defined in formula(I), and the chirality at R₃ is either R or S, or a mixture thereof,

or an activated derivative thereof with the amine HNR₄R₅, wherein R₄ andR₅ have the meaning defined in formula (I), under standard conditionsfor preparing amides from a carboxylic acid or an activated derivativethereof and an amine.

It will be appreciated that the mixture of diastereomers of compounds offormula (I) obtained from the above reaction may be separated usingstandard resolution techniques well known in the art, for example columnchromatography.

Thus the amide of formula (I) may be prepared by treating the carboxylicacid of formula (II) with an activating agent such as BOP(benzotriazol-1-yloxy-tris(dimethylamino)phosphoniumhexafluorophosphate), TBTU(2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate),BOP-Cl (bis(2-oxo-3-oxazolidinyl)phosphinic chloride), oxalyl chlorideor 1,1′-carbonyldiimidazole in an aprotic solvent such asdichloromethane optionally in the presence of a tertiary amine such astriethylamine and subsequent reaction of the product thus formed, i.e.the activated derivative of the compound of formula (II), with the amineHNR₄R₅.

Alternatively the amide of formula (I) may be prepared by reacting amixed anhydride derived from the carboxylic acid (II), wherein R₁, R₂and R₃ have the meanings defined in formula (I) with the amine HNR₄R₅ inan aprotic solvent such as tetrahydrofuran.

Conveniently the reaction is carried out at low temperatures, forexample 25° C. to −90° C., conveniently at approximately −78° C.

The mixed anhydride is conveniently prepared by reacting the carboxylicacid (II) with a suitable acid chloride e.g. pivalolyl chloride in anaprotic solvent such as ethyl acetate in the presence of a tertiaryorganic base such as a trialkylamine e.g. triethylamine and at lowtemperatures, for example 25° C. to −90° C., conveniently atapproximately −78° C.

Compounds of formula (I) may also be prepared by reacting a compound offormula (III)

wherein R₁, R₂ and R₃ have the meanings defined in formula (I) and R₆ is2-hydroxyphenyl, with 1,1′-carbonyldiimidazole or1,1′-thiocarbonyldiimidazole in a suitable solvent such asdichloromethane and subsequent reaction of the products thus formed withthe amine HNR₄R₅.

Compounds of formula (II) may be prepared from a compound of formula(III) wherein R₆ is 2-hydroxyphenyl by reaction with1,1′-carbonyldiimidazole or 1,1′-thiocarbonyldiimidazole in a suitablesolvent such as dichloromethane and subsequent reaction of the productthus formed with aqueous acetone.

Compounds of formula (III) wherein R₆ is 2-hydroxyphenyl may be preparedfrom the corresponding compounds of formula (III) wherein R₆ is a2-benzyloxyphenyl group by hydrogenolysis using hydrogen and a palladiumcatalyst.

Alternatively compounds of formula (III) wherein R₆ is a 2-hydroxyphenylmay be prepared from the compound of formula (IV)

wherein R₁, R₂ and R₃ have the meanings defined in formula (I), R₆ is2-benzyloxyphenyl, R₇ is benzyloxycarbonyl and R₈ is C₁₋₆alkyl, by thereaction with hydrogen in the presence of a palladium on charcoalcatalyst and acetic acid. This reaction is conveniently carried out in asolvent such as ethanol, trifluoroethanol or mixtures thereof.

Compounds of formula (IV) may be prepared by reacting the amino esterhydrochloride (V)

wherein R₁ has the meaning defined in formula (I) and R₈ is C₁₋₆alkyl,with an aldehyde R₃CHO (VI) wherein R₃ has the meaning defined informula (I), in the presence of triethylamine and in a solvent such astrifluoroethanol and then reacting the resultant product with a compoundof formula (VII)

wherein R₁ has the meaning defined in formula (I) and R₇ ist-butyloxycarbonyl or benzyloxycarbonyl and the isocyanide CNR₆ (VIII)wherein R₆ is a 2-benzyloxyphenyl group, in a solvent such astrifluoroethanol.

Compounds of formula (III) wherein R₆ is a 2-benzyloxyphenyl group maybe prepared from a compound of formula (IV) wherein R₁, R₂ and R₃ havethe meanings defined in formula (I), R₆ is 2-benzyloxyphenyl and R₇ ist-butyloxycarbonyl by the reaction with hydrogen chloride in dioxanfollowed with triethylamine in a solvent such as dichloromethane.

The compound of formula (IV) wherein R₇ is t-butyloxycarbonyl may beprepared by the route described above using a compound of formula (VII)wherein R₇ is t-butyloxycarbonyl.

The R₂ substituent is a 1-methylpropyl group and the compound of formula(I) wherein R₂ is a 1-methylpropyl group having an (S) or (R)configuration may be prepared by starting with the aminoesterhydrochloride (V) wherein the R₂ group has the required (S) or (R)configuration.

Aminoester hydrochloride (V), wherein R₁ has the meaning defined informula (I) and R₈ is C₁₋₆ alkyl, may be prepared from the correspondingcommercially available amino acids, D-alloisoleucine or D-isoleucine, bythe method of Schmidt, U; Kroner, M; Griesser, H. Synthesis (1989),(11), 832-5.

Aldehydes R₃CHO (VI), wherein R₃ has the meaning defined in formula (I),are either commercially available or may be prepared by literaturemethods (Comins, Daniel L.; Weglarz, Michael A.; J. Org. Chem.; 53; 19;1988; 4437-4442).

The aminoacid derivative (VII) wherein R₁ has the meaning defined informula (I) and R₇ is t-butyloxycarbonyl is commercially available; theaminoacid derivative (VII) wherein R₁ has the meaning defined in formula(I) and R₇ is benzyloxycarbonyl may be prepared from the correspondingcommercially available amino acid (R)—R₁CH(NH₂)CO₂H (IX), wherein R₁ hasthe meaning defined in formula (I), by treatment withN-(benzyloxycarbonyloxy)succinimde and triethylamine in a solvent suchas dioxane in water.

The isocyanide CNR₆ (VIII) may be prepared according to literaturemethods (Obrecht, Roland; Herrmann, Rudolf; Ugi, Ivar, Synthesis, 1985,4, 400-402).

Acid addition salts of the compound of formula (I) may be prepared byconventional means, for example, by treating a solution of the compoundin a suitable solvent such as dichloromethane or acetone, with asuitable solution of the appropriate inorganic or organic acid.

The following examples are illustrative, but not limiting of theembodiments of the present invention.

EXPERIMENTAL Nomenclature

All intermediates and examples were named using ACD Name Pro 6.02 inISISDraw.

Abbreviations

-   CV: Column volume. One column volume is defined as the volume    occupied by the sorbent in the packed column. This can be    approximately calculated from the mass and density of the particular    sorbent being used (1CV=mass divided by density).    General purification and analytical methods Analytical HPLC was    conducted on a Supelcosil LCABZ+PLUS column (3.3 cm×4.6 mm ID),    eluting with 0.1% HCO₂H and 0.01 M ammonium acetate in water    (solvent A), and 0.05% HCO₂H and 5% water in acetonitrile (solvent    B), using the either elution gradient 1, 0-0.7 minutes 0% B, 0.7-4.2    minutes 0%-100% B, 4.2-5.3 minutes 100% B, 5.3-5.5 minutes 0% B or    elution gradient 2, 0-0.7 minutes 0% B, 0.7-4.2 minutes 0%-100% B,    4.2-4.6 minutes 100% B, 4.6-4.8 minutes 0% B at a flow rate of 3    ml/minute. Retention times (Rt) are quoted in minutes. The mass    spectra (MS) were recorded on a Waters ZQ 2000 mass spectrometer    using electrospray positive [ES+ve to give MH⁺ and M(NH₄)⁻ molecular    ions] or electrospray negative [ES-ve to give (M−H)⁻ molecular ion]    modes. ¹H NMR spectra were recorded using a Bruker DPX 400 MHz    spectrometer using tetramethylsilane as the external standard.

Purification using silica cartridges refers to chromatography carriedout using a Combiflash® Companion™ with Redisep® cartridges supplied byPresearch. Hydrophobic frits refer to filtration tubes sold by Whatman.SPE (solid phase extraction) refers to the use of cartridges sold byInternational Sorbent Technology Ltd. TLC (thin layer chromatography)refers to the use of TLC plates sold by Merck coated with silica gel 60F₂₅₄.

Intermediate 1 MethylN-[(2R)-2-(2,3-dihydro-1H-inden-2-yl)-2-({[(phenylmethyl)oxy]carbonyl}amino)acetyl]-N-[1-(2,6-dimethyl-3-pyridinyl)-2-oxo-2-({2-[(phenylmethyl)oxy]phenyl}amino)ethyl]-D-alloisoleucinate

2,6-dimethylpyridine-3-carboxaldehyde (Aurora Feinchemie GmbH) (2.00 g,16.1 mmol) and (D)-alloisoleucine methyl ester hydrochloride (2.93 g,16.1 mmol) in methanol (50 mL) and 2,2,2-trifluoroethanol (50 mL) weretreated with triethylamine (2.24 mL, 16.1 mmol) and the mixture wasstirred under nitrogen at room temperature for 20 h.(2R)-2,3-dihydro-1H-inden-2-yl({[(phenylmethyl)oxy]carbonyl}amino)ethanoicacid (5.24 g, 16.1 mmol) and 2-benzyloxyphenylisonitrile (3.37 g, 16.1mmol) were added and the mixture was stirred at room temperature undernitrogen for 4 days. The mixture was concentrated under reduced pressurethen partitioned between ethyl acetate (150 mL) and water (150 mL) plussaturated aqueous sodium hydrogen carbonate (6 mL). The aqueous phasewas back-extracted with ethyl acetate (50 mL) and the combined organicextracts were washed successively with semi-saturated aqueous solutionsof sodium hydrogen carbonate, ammonium chloride and sodium chloride (100mL each), dried over anhydrous magnesium sulphate and evaporated underreduced pressure to give the crude product (12.01 g). This was purifiedon a Redisep silica column (330 g) eluted with 20-50% ethyl acetate incyclohexane to afford 7.46 g of the title compound as a pair ofdiastereomers.

HPLC Rt=3.88 and 3.96 minutes (gradient 1); m/z [M+H]⁺=797

Intermediate 22-{(3R,6R)-3-(2,3-dihydro-1H-inden-2-yl)-6-[(1S)-1-methylpropyl]-2,5-dioxo-1-piperazinyl}-2-(2,6-dimethyl-3-pyridinyl)-N-(2-hydroxyphenyl)acetamide

The crude methylN-[(2R)-2-(2,3-dihydro-1H-inden-2-yl)-2-({[(phenylmethyl)oxy]carbonyl}amino)acetyl]-N-[1-(2,6-dimethyl-3-pyridinyl)-2-oxo-2-({2-[(phenylmethyl)oxy]phenyl}amino)ethyl]D-alloisoleucinate(intermediate 1) (7.46 g) was dissolved in ethanol (150 mL) and aceticacid (10 mL) and the mixture was hydrogenated at 1 atmosphere of H₂ over10% palladium on carbon (Degussa type) (1.8 g wetted with water 1:1 w:w)for 18 h. The reaction mixture was evaporated under reduced pressure andthe residue was partitioned between ethyl acetate and water withsaturated aqueous sodium hydrogen carbonate added until the aqueousphase was basic (pH 8). The aqueous phase was extracted with ethylacetate and the combined organic extracts were washed with saturatedaqueous sodium hydrogen carbonate:water 3:1 (100 mL) then with saturatedbrine before being dried over anhydrous magnesium sulphate andevaporated under reduced pressure. The crude product was purified on aRedisep silica column (120 g) eluted with 0-10% methanol in ethylacetate to give the title compound as a pair of diastereomers (2.94 g).

HPLC Rt=2.75 and 2.81 minutes (gradient 2); m/z [M+H]⁺=541

Intermediate 32-{(3R,6R)-3-(2,3-Dihydro-1H-inden-2-yl)-6-[(1S)-1-methylpropyl]-2,5-dioxo-1-piperazinyl}-2-(4,6-dimethyl-3-pyridinyl)-N-{2-[(phenylmethyl)oxy]-phenyl}acetamide

4,6-Dimethyl-3-pyridinecarbaldehyde¹ (2.52 g) and methylD-alloisoleucinate hydrochloride (3.4 g) were dissolved in2,2,2-trifluoroethanol (50 ml). To this was added triethylamine (2.61ml) and the reaction mixture was left to stand for 18 hours.(2R)-2,3-Dihydro-1H-inden-2-yl({[(1,1-dimethylethyl)oxy]-carbonyl}amino)ethanoicacid (5.44 g) and 2-[(phenylmethyl)oxy]phenyl isocyanide (4.18 g) withmethanol (10 ml) were added to the reaction mixture and the solution wasstirred at room temperature for 3 days. The solvent was removed in vacuoand the residue was separated between dichloromethane and water. Theorganic phase was passed through a hydrophobic frit and evaporated invacuo. The residue was dissolved in 4N hydrogen chloride in dioxan (50ml) and the reaction mixture was left to stand for 4 hours. The solventwas removed in vacuo and the residue was dissolved in dichloromethane(200 ml). To this was added triethylamine (20 ml) and the reactionmixture was left to stand for 20 hours. The reaction mixture wasseparated between dichloromethane and water. The organic phase waspassed through a hydrophobic frit and evaporated in vacuo. The residuewas applied to 4×90 g Biotage columns and eluted with cyclohexane/ethylacetate (1:1, 1:2 v/v) and ethyl acetate. The required fractions werecombined and evaporated in vacuo to give2-{(3R,6R)-3-(2,3-dihydro-1H-inden-2-yl)-6-[(1S)-1-methylpropyl]-2,5-dioxo-1-piperazinyl}-2-(4,6-dimethyl-3-pyridinyl)-N-{2-[(phenylmethyl)oxy]phenyl}acetamide(5.55 g, 47%) as a tan foam.

HPLC Rt=3.43, 3.45 minutes gradient 1); m/z [M+H]⁺=631

Ref:

-   1. Comins, Daniel L.; Weglarz, Michael A.; J. Org. Chem.; 53; 19;    1988; 4437-4442.

Intermediate 42-{(3R,6R)-3-(2,3-Dihydro-1H-inden-2-yl)-6-[(1S)-1-methylpropyl]-2,5-dioxo-1-piperazinyl}-2-(4,6-dimethyl-3-pyridinyl)-N-(2-hydroxyphenyl)acetamide

2-{(3R,6R)-3-(2,3-Dihydro-1H-inden-2-yl)-6-[(1S)-1-methylpropyl]-2,5-dioxo-1-piperazinyl}-2-(4,6-dimethyl-3-pyridinyl)-N-{2-[(phenylmethyl)oxy]phenyl}acetamide(intermediate 3) (3.30 g) was dissolved in ethanol (75 ml) andhydrogenated over palladium on charcoal (wet 10% Pd, 0.50 g) for 20hours. The catalyst was removed by filtration and washed withdichloromethane. The combined filtrate and washings were evaporated invacuo. The residue was applied to a 90 g Biotage column and eluted withethyl acetate. The required fractions were combined and evaporated invacuo to give2-{(3R,6R)-3-(2,3-dihydro-1H-inden-2-yl)-6-[(1S)-1-methylpropyl]-2,5-dioxo-1-piperazinyl}-2-(4,6-dimethyl-3-pyridinyl)-N-(2-hydroxyphenyl)acetamide(2.43 g, 87%) as a pale yellow solid.

HPLC Rt=2.86 minutes (gradient 1); m/z [M+H]⁺=541.

Intermediate 5{(3R,6R)-3-(2,3-Dihydro-1H-inden-2-yl)-6-[(1S)-1-methylpropyl]-2,5-dioxo-1-piperazinyl}(2,6-dimethyl-3-pyridinyl)aceticacid hydrochloride

2-{(3R,6R)-3-(2,3-dihydro-1H-inden-2-yl)-6-[(1S)-1-methylpropyl]-2,5-dioxo-1-piperazinyl}-2-(2,6-dimethyl-3-pyridinyl)-N-(2-hydroxyphenyl)acetamide(24.25 g, 45 mmol) (intermediate 2) and 1,1′-carbonyldiimidazole (11.7g, 72 mmol) were dissolved in dry dichloromethane (200 ml) and left tostand under nitrogen for 20 hours. The solvent was removed in vacuo andthe residue was dissolved in acetone (200 ml) and 2N hydrochloric acid(20 ml). After stirring for 20 hours the solvent was removed in vacuoand the residue was dissolved in methanol (50 ml). The solution wasapplied to an aminopropyl cartridge (2×70 g) and eluted with methanol(250 ml) and then 10% acetic acid in methanol (250 ml). The requiredfractions were combined and evaporated in vacuo. The residue was treatedwith 2N hydrochloric acid and the resulting solution evaporated in vacuoto give the title compound{(3R,6R)-3-(2,3-dihydro-1H-inden-2-yl)-6-[(1S)-1-methylpropyl]-2,5-dioxo-1-piperazinyl}(2,6-dimethyl-3-pyridinyl)aceticacid hydrochloride as a tan solid (12.21 g, 56%).

HPLC Rt=2.48 minutes (gradient 2); m/z [M+H]⁺=450.

Intermediate 6(2R)-2,3-dihydro-1H-inden-2-yl({[(phenylmethyl)oxy]carbonyl}amino)ethanoicacid

(2R)-amino(2,3-dihydro-1H-inden-2-yl)ethanoic acid (1.91 g, 10 mmol) wassuspended in dioxane (10 ml) and water (10 ml). To this was addedtriethylamine (1.7 ml) and N-(benzyloxycarbonyloxy)-succinimde (2.54 g)and the reaction mixture was stirred rapidly at room temperature for 2days. The reaction mixture was poured into water (50 ml) and extractedwith chloroform (100 ml). The organic phase was washed with 1Nhydrochloric acid (50 ml) and water (50 ml). This was dried overmagnesium sulphate and the solvent removed in vacuo to give the titlecompound (3.06 g, 94%):

¹H NMR (CDCl₃) δ 7.40-7.29 (m, 5H), 7.21-7.11 (m, 4H), 5.28 (d, 1H,J=8.6 hZ), 5.11 (s, 2H), 4.57 (m, 1H), 3.14-2.79 (m, 5H); LCMS m/z 326(MH⁺), Rt 3.35 min (gradient 2)

Example 1

(2R)-2-{(3R,6R)-3-(2,3-Dihydro-1H-inden-2-yl)-6-[(1S)-1-methylpropyl]-2,5-dioxo-1-piperazinyl}-2-(2,6-dimethyl-3-pyridinyl)-N-methylethanamide

2-{(3R,6R)-3-(2,3-dihydro-1H-inden-2-yl)-6-[(1S)-1-methylpropyl]-2,5-dioxo-1-piperazinyl}-2-(2,6-dimethyl-3-pyridinyl)-N-(2-hydroxyphenyl)acetamide(intermediate 2) (0.400 g, 0.74 mmol) and 1,1′-carbonyldiimidazole(0.192 g, 1.18 mmol) in dry dichloromethane (10 mL) were stirred at roomtemperature under N₂ for 7 hrs. The mixture was treated with a 2Msolution of methylamine in tetrahydrofuran (1.849 mL, 3.70 mmol) andleft to stand overnight at room temperature. The solvents were blowndown under N₂ and the residue was purified on a Redisep silica column(35 g) eluted with 0-10% methanol in ethyl acetate followed by furtherpurification on a Kromasil KR100-10-C18 reverse-phase column eluted withaqueous acetonitrile (20-45% MeCN) containing 0.1% formic acid. Thisgave the title compound as a white lyophilisate (30%) afterfreeze-drying from 1,4-dioxane.

HPLC Rt=2.44 minutes (gradient 1); m/z [M+H]⁺=463

¹H NMR (CDCl₃) δ 7.63 (d, 1H), 7.25-7.15 (m, 4H), 7.05 (d, 1H), 6.79 (d,1H), 5.96 (q, 1H), 5.35 (s, 1H), 4.07 (dd, 1H), 3.88 (d, 1H), 3.19-2.88(m, 4H), 2.85 (d, 3H), 2.81-2.73 (m, 1H), 2.56 (s, 3H), 2.55 (s, 3H),1.82-1.67 (m, 2H), 1.20-1.08 (m, 1H), 0.99 (d, 3H), 0.90 (t, 3H).

Similarly prepared from intermediate 2 and dimethylamine (2.0M intetrahydrofuran):

Example 2

(2R)-2-{(3R,6R)-3-(2,3-Dihydro-1H-inden-2-yl)-6-[(1S)-1-methylpropyl]-2,5-dioxo-1-piperazinyl}-2-(2,6-dimethyl-3-pyridinyl)-N,N-dimethylethanamideas a white lyophilisate (33%) after freeze-drying from 1,4-dioxane

HPLC Rt=2.69 minutes (gradient 1); m/z [M+H]⁺=477

¹H NMR (CDCl₃) δ 7.49 (d, 1H), 7.27-7.15 (m, 4H), 7.08 (d, 1H), 6.66 (s,1H), 6.30 (d, 1H), 4.10 (dd, 1H), 4.05 (d, 1H), 3.22-3.08 (m, 3H),2.99-2.84 (m, 4H), 2.80-2.70 (m, 4H), 2.63 (s, 3H), 2.58 (s, 3H),1.65-1.53 (m, 1H), 0.97-0.78 (m, 2H), 0.71 (t, 3H), 0.46 (d, 3H).

Similarly prepared from intermediate 2 and morpholine (3.7 mmol):

Example 3 (Method A)

(3R,6R)-3-(2,3-dihydro-1H-inden-2-yl)-1-[1R)-1-(2,6-dimethyl-3-pyridinyl)-2-(4-morpholinyl)-2-oxoethyl]-6-[(1S)-1-methylpropyl]-2,5-piperazinedioneas a white lyophilisate (88 mg, 23%) after freeze-drying from1,4-dioxane

HPLC Rt=2.70 minutes (gradient 2); m/z [M+H]⁺=519

¹H NMR (CDCl₃) δ 7.49 (d, 1H), 7.27-7.15 (m, 4H), 7.10 (d, 1H), 6.68 (s,1H), 6.40 (d, 1H), 4.10 (dd, 1H), 4.01 (d, 1H), 3.74-3.52 (m, 5H),3.28-3.07 (m, 5H), 2.97-2.84 (m, 2H), 2.79-2.71 (m, 1H), 2.62 (s, 3H),2.59 (s, 3H), 1.65-1.53 (m, 1H), 0.98-0.80 (m, 2H), 0.70 (t, 3H), 0.45(d, 3H).

Example 3 (Method B)

(3R,6R)-3-(2,3-Dihydro-1H-inden-2-yl)-1-[(1R)-1-(2,6-dimethyl-3-pyridinyl)-2-(4-morpholinyl)-2-oxoethyl]-6-[(1S)-1-methylpropyl]-2,5-piperazinedione

A suspension of{(3R,6R)-3-(2,3-dihydro-1H-inden-2-yl)-6-[(1S)-1-methylpropyl]-2,5-dioxo-1-piperazinyl}(2,6-dimethyl-3-pyridinyl)aceticacid hydrochloride (5.0 g, 10.3 mmol) (intermediate 5) in drydichloromethane (50 ml) was treated with 1,1-carbonyldiimidazole (2.6 g,16 mmol) and the reaction mixture was stirred under nitrogen for 18hours. Morpholine (4.8 ml, 55 mmol) was added and the resultant solutionwas left to stand under nitrogen for 18 hours. The solvent was removedin vacuo and the residue was separated between ethyl acetate and water.The organic phase was washed with brine and dried over anhydrousmagnesium sulphate. The solvent was removed in vacuo and the residue wasdissolved in dichloromethane. This was applied to a basic aluminacartridge (240 g) and eluted using a gradient of 0-7.5% methanol indiethyl ether (9CV), 7.5-10% methanol in diethyl ether (1CV) and 10%methanol in diethyl ether (1CV). The required fractions were combinedand evaporated in vacuo to give(3R,6R)-3-(2,3-dihydro-1H-inden-2-yl)-1-[(1R)-1-(2,6-dimethyl-3-pyridinyl)-2-(4-morpholinyl)-2-oxoethyl]-6-[(1S)-1-methylpropyl]-2,5-piperazinedioneas a white solid (2.4 g, 45%).

HPLC Rt=2.72 minutes (gradient 2); m/z [M+H]⁺=519

Example 4

(2R)-2-{(3R,6R)-3-(2,3-Dihydro-1H-inden-2-yl)-6-[(1S)-1-methylpropyl]-2,5-dioxo-1-piperazinyl}-2-(4,6-dimethyl-3-pyridinyl)-N,N-dimethylethanamide

2-{(3R,6R)-3-(2,3-Dihydro-1H-inden-2-yl)-6-[(1S)-1-methylpropyl]-2,5-dioxo-1-piperazinyl}-2-(4,6-dimethyl-3-pyridinyl)-N-(2-hydroxyphenyl)acetamide(intermediate 4) (0.700 g) and 1,1′-carbonyldiimidazole (0.324 g) weredissolved in dry dichloromethane (20 ml) and left to stand for 20 hours.To one half of this solution (10 ml) was added a 2.0M solution ofdimethylamine in tetrahydrofuran (5 ml) and the reaction mixture wasleft to stand for 3 days. The reaction mixture was separated betweendichloromethane and saturated aqueous sodium bicarbonate solution. Theorganic phase was passed through a hydrophobic frit and evaporated invacuo. The residue was applied to a silica cartridge (10 g) and elutedwith ethyl acetate then 5% methanol in ethyl acetate. The requiredfractions were evaporated in vacuo and the residue was purified furtherusing Mass Directed AutoPrep. This gave(2R)-2-{(3R,6R)-3-(2,3-Dihydro-1H-inden-2-yl)-6-[(1S)-1-methylpropyl]-2,5-dioxo-1-piperazinyl}-2-(4,6-dimethyl-3-pyridinyl)-N,N-dimethylethanamide(0.10 g, 32%) as a white foam.

HPLC Rt=2.82 minutes (gradient 1); m/z [M+H]⁺=477

¹H NMR (CDCl₃) δ 8.32 (s, 1H), 7.26-7.15 (m, 4H), 7.08 (s, 1H), 6.71 (s,1H), 6.16 (d, 1H), 4.17 (d, 1H), 4.10 (dd, 1H), 3.22-3.06 (m, 3H), 2.98(s, 3H), 2.91 (m, 1H), 2.74 (dd, 1H), 2.67 (s, 3H), 2.57 (s, 3H), 2.39(s, 3H), 1.56 (m, 1H), 0.93 (m, 1H), 0.85 (m, 1H), 0.68 (t, 3H), 0.45(d, 3H).

Similarly prepared from intermediate 4 and methylamine:

Example 5(2R)-2-{(3R,6R)-3-(2,3-Dihydro-1H-inden-2-yl)-6-[(1S)-1-methylpropyl]-2,5-dioxo-1-piperazinyl}-2-(4,6-dimethyl-3-pyridinyl)-N-methylethanamide

HPLC Rt=2.60 minutes (gradient 1); m/z [M+H]⁺=463

¹H NMR (CDCl₃) δ 8.48 (s, 1H), 7.25-7.14 (m, 4H), 7.04 (s, 1H), 6.72 (d,1H), 6.07 (q, 1H), 5.45 (s, 1H), 4.07 (dd, 1H), 3.90 (d, 1H), 3.17-3.04(m, 3H), 2.92 (m, 1H), 2.86 (d, 3H), 2.76 (dd, 1H), 2.53 (s, 3H), 2.33(s, 3H), 1.70 (m, 2H), 1.12 (m, 1H), 0.94 (d, 3H), 0.87 (t, 3H).

Similarly prepared from intermediate 4 and morpholine:

Example 6

(3R,6R)-3-(2,3-dihydro-1H-inden-2-yl)-1-[(1R)-1-(4,6-dimethyl-3-pyridinyl)-2-(4-morpholinyl)-2-oxoethyl]-6-[(1S)-1-methylpropyl]-2,5-piperazinedione

HPLC Rt=2.94 minutes (gradient 2); m/z [M+H]⁺=519

¹H NMR (CDCl₃) δ 8.34 (s, 1H), 7.25-7.15 (m, 4H), 7.09 (s, 1H), 6.71 (s,1H), 6.21 (d, 1H), 4.14-4.07 (m, 2H), 3.73-3.47 (m, 5H), 3.23-3.05 (m,5H), 2.95-2.83 (m, 2H), 2.74 (dd, 1H), 2.58 (s, 3H), 2.38 (s, 3H), 1.56(m, 1H), 0.94 (m, 1H), 0.86 (m, 1H), 0.68 (t, 3H), 0.44 (d, 3H).

Biological Activity

Examples 1-6 of the present invention were tested in all of the assaysdescribed below. Results for each of the compounds are shown in Table 1below. Table 1 also includes a compound X for comparison.

Assay 1 Determination of Antagonist Affinity at Human Oxytocin-1Receptors Using FLIPR Cell Culture

Adherent Chinese Hamster Ovary (CHO) cells, stably expressing therecombinant human Oxytocin-1 (hOT) receptor, were maintained in culturein DMEM:F12 medium (Sigma, cat no D6421), supplemented with 10% heatinactivated foetal calf serum (Gibco/Invitrogen, cat. no. 01000-147), 2mM L-glutamine (Gibco/Invitrogen, cat. no. 25030-024) and 0.2 mg/ml G418(Gibco/Invitrogen, cat no. 10131-027). Cells were grown as monolayersunder 95%:5% air:CO₂ at 37° C. and passaged every 3-4 days using TrypLE™Express (Gibco/Invitrogen, cat no. 12604-013).

Measurement of [Ca²⁺]_(i) Using the FLIPR™

CHO-hOT cells were seeded into black walled clear-base 384-well plates(Nunc) at a density of 10,000 cells per well in culture medium asdescribed above and maintained overnight (95%:5% air:CO₂ at 37° C.).After removal of culture medium, cells were incubated for 1 h at 37° C.in Tyrode's medium (NaCl, 145 mM; KCl, 2.5 mM; HEPES, 10 mM; Glucose, 10mM; MgCl₂, 1.2 mM; CaCl₂, 1.5 mM) containing probenacid (0.7 mg/ml), thecytoplasmic calcium indicator, Fluo-4 (4 uM; Teflabs, USA) and thequenching agent Brilliant Black (250 uM; Molecular Devices, UK). Cellswere then incubated for an additional 30 min at 37° C. with eitherbuffer alone or buffer containing OT antagonist, before being placedinto a FLIPR™ (Molecular Devices, UK) to monitor cell

fluorescence (λ_(ex)=488 nm, λ_(EM)=540 nm) before and after theaddition of a submaximal concentration of oxytocin (EC80).

Data Analysis

Functional Responses Using FLIPR were Analysed Using Activity BaseVersion 5.0.10.

Assay 2 Oxytocin Binding Assay Preparations

Membranes were prepared from CHO cells expressing human recombinantoxytocin receptors. The membrane preparation was frozen in aliquots at−70° C. until used.

Binding Assay Protocol

Membranes (˜50 ug) were incubated in 200 ul of assay buffer (50 mM Tris,10 mM MgCl₂, and 0.1% bovine serum albumin, pH 7.5) containing −2.4 nMof [3H]-oxytocin in the absence (total binding) or presence(non-specific binding) of 1 uM unlabeled oxytocin and increasingconcentrations of the compounds in Examples 1 to 6 or comparatorcompounds. Incubations were performed at room temperature for 60minutes. The reactions were stopped with 3 ml of ice cold buffer andfiltered through

Whatman GF/C filter paper presoaked in 0.3% polyethylenimine. Thefilters were washed 4 times with 3 ml buffer using a Brandel cellharvester. The filters were counted in 3 ml Ready Safe scintillationfluid (Beckman).

Specific binding represented approximately 90% of total binding.

Data Analysis

IC₅₀ values were determined from competition binding experiments usingnon-linear regression analysis (GraphPad) and converted to Ki using themethod of Cheng and Prusoff, 1974. Data are reported as mean values.

Assay 3

Determination of In vitro Intrinsic Clearance in Microsomes

NADP regeneration buffer for use in incubations was prepared fresh onthe assay day. It contained 7.8 mg glucose-6-phosphate (mono-sodiumsalt), 1.7 mg NADP and 6 Units glucose-6-phosphate dehydrogenase per 1mL of 2% sodium bicarbonate. Microsomes (human, female; cynomolgusmonkey, female; dog, female; rat, female) were prepared in pH7.4phosphate buffer and contained 0.625 mg protein/mL. Unless stated, allsubsequent steps were performed by a Tecan Genesis 150/8 RSP. A 1.25 mMstock solution of the compounds was prepared in Acetonitrile/water(1:1). 25 ul of the 1.25 mM stock solution was added to 600 ul ofAcetonitrile/water (1:1) to give a 50 uM solution. For each species, the50 uM solutions (10 uL) were added to microsomes (790 uL) in amicroplate (Porvair, 96 deepwell, square). 400 uL of the microsomalsolution containing the compound was transferred to a microplate(Porvair, 96 deepwell, round) and was pre-warmed at 37° C. for fiveminutes prior to initiation of incubations. All incubations wereinitiated by addition of 100 uL of NADP regeneration system to thepre-warmed microsomes. The mixtures were incubated at 37° C. in a Techneheating block. Following 0, 3, 6, 12 and 30 minutes incubation, 20 uLaliquots were taken and added to 100 uL of acetonitrile containinginternal standard.

For determination of the rate of metabolism, incubations were performedat a compound concentration of 0.5 uM and a protein concentration of 0.5mg/mL. The concentration of solvent in the incubation was 0.5%.

Test compound concentrations were determined by LC/MS/MS; results werereported as analyte:internal standard peak area ratios.

The rate of disappearance was calculated by fitting a single exponentialdecay to the concentration-time curve using Excel and intrinsicclearance was calculated using the following formula:

${Cli} = \frac{\left\lbrack {{{rate}\left( {1\text{/}\min} \right)}*52.5\mspace{14mu} {mg}\mspace{14mu} {protein}\text{/}g\mspace{14mu} {liver}} \right\rbrack}{0.5\mspace{14mu} {mg}\mspace{14mu} {protein}\text{/}{mL}}$

Results

Examples 1 to 6 of the present invention and also a comparator compoundX=(2R)-2-[(3R,6R)-3-(2,3-dihydro-1H-inden-2-yl)-6-isobutyl-2,5-dioxopiperazin-1-yl]-N,N-dimethyl-2-(6-methylpyridin-3-yl)ethanamide(Example 209 in WO 03/053443) were tested in the above assays.

Comparator compound X when tested in assays 1 and 2 showed a similarpotency to that exhibited by Compounds 1 to 6 of the present invention,in fact each of these compounds exhibited fpKi's of between 8.1 and 9.2(Assay 1) and pKi's of between 8.8 and 10.5 (Assay 2).

However, the compounds of the present invention exhibited a surprisingimprovement in in vitro intrinsic clearance in microsomes (Assay 3) whencompared with comparator compound X.

TABLE 1 Microsomal CI (ml/min/g) Assay 3 Rat Dog Cynomolgus monkey HumanComparator X + + ++++ ++ Example 1 + + + + Example 2 + + ++, +++ +, +Example 3 + + + + Example 4 +, + +, + +++++, +++++ ++, + Example 5 + ++++ + Example 6 + + +++ + Key to Table 1 + corresponds to 1-8 ml/min/mg++ corresponds to 9-15 ml/min/mg +++ corresponds to 16-20 ml/min/mg ++++corresponds to 21-30 ml/min/mg +++++ corresponds to >31 ml/min/mg

1. A process for the preparation of compounds of formula (I)

wherein: R₁ is 2-indanyl, R₂ is 1-methylpropyl, R₃ is a group selectedfrom 2,6-dimethyl-3-pyridyl and 4,6-dimethyl-3-pyridyl, R₄ is methyl,and R₅ represents hydrogen or methyl, or R₄ and R₅ together with thenitrogen atom to which they are attached represent morpholino; where themethod comprises reacting a compound of formula (III)

wherein R₆ is 2-hydroxyphenyl, with 1,1′-carbonyldiimidazole or1,1′-thiocarbonyldiimidazole in a suitable solvent and subsequentreaction of the product thus formed with amine HNR₄R₅.